Angiogenesis refers to the new blood vessels formed from the development of pre-existing capillaries or post-capillary venules, which is a complex process involving many molecules of multiple cells. Angiogenesis is a complex process of coordination of angiogenic factors and inhibitory factors, when are in a state of balance under normal condition. The vascular system would be activated once the balance is broken, resulting in excessive angiogenesis or vascular degeneration by inhibition of the vascular system.
There are a number of diseases known to be associated with deregulated or undesired angiogenesis. Such diseases include, but are not limited to, tumors for example so-called solid tumors and liquid (or hematopoietic) tumors (e.g. leukemias and lymphomas), inflammatory diseases e.g. rheumatoid or rheumatic inflammation, especially arthritis (including rheumatoid arthritis), or other chronic inflammation e.g. chronic asthma, arteriosclerosis or post transplant arteriosclerosis, endometriosis, ocular neovascular diseases such as retinopathies (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis. Other diseases associated with deregulated or undesired angiogenesis will be apparent to those skilled in the art.
Vascular endothelial growth factor (VEGF), a heparin-binding growth factor specific for the vascular endothelial cell, may induce angiogenesis in vivo. It includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental growth factor.
The main function of VEGF-A is to facilitate proliferation and migration of the vascular endothelial cell, and lumen formation, as well as to increase vascular leakage, promote the chemotaxis of monocytes and the formation of B cells. The biological effects of VEGF-A are mediated by the binding with its specific receptor, which mainly includes the specific receptors of vascular endothelial growth factor receptor 1 (VEGFR-1) and VEGFR-2. Moreover, VEGFR-2 is considered to be the major VEGFR, which exerts significant effect on proliferation of the vascular endothelial cell. VEGFR-2 induces, by intracellular kinase, VEGF to bind to the dimer and the receptors to be autophosphorylated, thereby enhancing cell mitosis (Klettner A, Roider J. Treating age-related macular degeneration interaction of VEGF-antagonists with their target. Mini Rev Med Chem, 2009, 9 (9): 1127-1135). VEGF-A comprising eight exons and seven introns is spliced into several isoforms during transcription, mainly including VEGF121, VEGF145, VEGF206, VEGF165 and VEGF189, which are of different molecular weight, solubility and heparin-binding capability. Among them, VEGF165 is the most predominant isoform of VEGF-A (Ferrara N, Gerber H P, Le Couter J. The biology of VEGF and its receptors, Nat Med. 2003, 9 (6): 669-676). VEGF165, which is a secreted soluble protein, directly acts on the vascular endothelial cell, thereby promoting cell proliferation, accelerating cell damage repair, increasing vascular permeability, reducing the intravascular thrombosis and occlusion of thrombus, and restraining intimal hyperplasia (Huang Chen-xing, Shen Zu-guang, Vascular endothelial growth factor—fundamental research and experimental study in plastic surgery, Chinese J Reparative and Reconstructive Surgery, 2002, 160: 64-68).
The existing drugs against vascular endothelial growth factor include Pegaptanib sodium (Macugen™), Ranibizumab (Lucentis™), Bevacizumab (Avastin™), VEGF Trap, etc. Currently, the controversy over anti-VEGF agents focuses on the possible exacerbation of the formation of tissue fibrous membranes. A major problem for the current anti-VEGF agents for the clinical treatment of various diseases (such as age-related macular degeneration) is that the agents have to be intraocularly injected frequently, thus inducing a potential risk for endophthalmitis. Researchers observed the effect of Bevacizumab and Macugen on different VEGF isoforms using the umbilical vein endothelial cell and Tenon fibrocyte. The results revealed that VEGF-165 and VEGF-121 mainly affect the angiogenesis, while VEGF-189 mainly affects the fibrogenesis process. Bevacizumab and Lucentis can inhibit all of the active isoforms of VEGF-A (Van Bergen T, Vandewalle E, Van de Veire S, et al. The role of different VEGF isoforms in scar formation after glaucoma filtration surgery. Exp Eye Res, 2011, 93: 689-699; and CATT research group, Martin D F, Maguire M G, et al. Ranibizumab and Bevacizumab for neovascular age-related macular degeneration. N Engl J Med, 2011. 364: 1897-1908), which might be the reason why Bevacizumab induced fibrosis of the vitreous cavity in some patients.
Currently, treatment using anti-VEGF agents has to be repeated every 4-6 week, while the annual average injection for Lucentis treatment in the 1st year is about 6.9 times, and for Bevacizumab treatment, about 7.7 times (Li X, Hu Y, Sun X, Zhang J, Zhang M. Bevacizumab for neovascular age-related macular degeneration in China. Ophthalmology. 2012 October, 119(10): 2087-93). Due to the frequent intraocular injection, there is a potential risk for endophthalmitis. Consequently, the development of a novel antibody drug with long-lasting efficacy as well as better retinal absorption and permeability is urgently needed, so as to prolong the administration cycle and to reduce the discomfort and risk resulting from the injection in the patient.
In addition, the current techniques for expression and purification of anti-VEGF agents are complex, and in most cases entail high cost, poor stability and limited application.
A heavy chain antibody is an antibody isolated from the serum of a camel, which consists exclusively of heavy chains. Its antigen binding region is merely a single-domain connected to Fc region via a hinge region, and the antigen binding region still maintains the antigen binding function upon removal from the antibody. Thus the heavy chain antibody is also known as single domain antibody (sdAb) or nano antibody (nanobody). Unlike the conventional antibody, the single-domain antibody is a peptide chain of about 110 amino acids, with a molecular weight 1/10 of a conventional antibody, which provides a new way for the molecular construction of an antibody (Muyldermans, Single domain camel antibodies: current status. J Biotechnol 2001, 74:277-302). This type of single-domain antibody with a small molecule weight is heat-stable and also stable towards the detergent and high concentration of uric acid, with superior tissue permeability in vivo and enhanced solubility (Stanfield R, Dooley H, Flajnik M, Wilson I. Crystal structure of a shark single-domain antibody V region in complex with lysozyme. Science. 2004, 305 (5691)). The single-domain antibody facilitates expression and is suitable for expression in a prokaryotic system. The single-domain antibody is further characterized by, e.g., low production cost, unique antigen recognition epitope, and the capability of identifying hidden antigenic sites. As a result, the single-domain antibody plays an increasingly and unimaginably huge role in terms of immunological assay, diagnosis and treatment (Dirk Saerens, Gholamreza Hassanzadeh Ghassabeh, Serge Muyldermans. Single-domain antibodies as building blocks for novel therapeutics. Current Opinion in Pharmacology 2008, 8:600-608).
Therefore, an antibody which is able to overcome the above mentioned problems from the existing anti-VEGF agents is needed in the art, for example, a single domain antibody which can specifically bind VEGF and inhibit the activity thereof.